Anti-angiogenic therapy promises to be a powerful tool for the treatment of solid tumors. The attraction of this therapy is that drug resistance is unlikely to develop and that it is likely to be effective in a broad range of tumor types. Limitations, at least for use of anti-angiogenic proteins, are that delivery cannot be oral and large amounts are currently needed for efficacy. Furthermore, little is known about the mechanism of action of these agents. This proposal focuses on endostatin, a powerful anti-angiogenic protein, and addresses some of these limitations. In Preliminary Studies, the PI has produced a soluble version of mouse endostatin in yeast, shown that the protein is active with high specific activity in proliferation and migration assays, in the CAM assay and in a xenograft renal cancer model. He has also generated a neutralizing antibody and discovered slightly differing structural mutants of endostatin with markedly contrasting activity, thus confirming that endostatin, and not a contaminant, is the active anti-angiogenic agent. Effects of endostatin on two intracellular signaling events mediated by vascular endothelial growth factor (VEGF), a pro-angiogenic agent have been delineated, and data is presented on endostatin's effect on the cell cycle. This proposal is a logical extension of these observations: Specific Aim 1: Signaling: cell cycle and apoptotic effects; cell surface events and downstream intracellular pathways Specific Aim 2: Mutants: structure-function analysis of endostatin In addition to furthering our understanding of endothelial cell biology, the findings in this proposal are likely to have major therapeutic implications: a delineation of endostatin's active moiety and an understanding of its mechanism of action will allow for its optimal and rational use in tumor therapy, and other angiogenesis dependent disease states as well.